ABSTRACT Interactions of alcohol with drugs, including the drugs of abuse, constitute a significant health problem. The mechanisms of these effects are tightly related to the influence of alcohol on drug?metabolizing system, and on cytochromes P450 in particular. The role of alcohol?metabolizing and alcohol?inducible cytochrome P450 2E1 (CYP2E1) in alcohol?induced oxidative stress and alcohol?induced liver injury is well recognized. However, CYP2E1 is not functional as a stand?alone enzyme, but rather constitutes a part of a complex multienzyme system. Liver cells possess an ensemble of multiple drug?metabolizing cytochromes P450 that are co?localized in the membrane of endoplasmic reticulum, share common redox partners and interact with each other with the formation of heteromeric complexes. According to the current concepts, these interactions considerably change the functional parameters of the individual P450 enzymes, so that the changes in the content of any particular P450 enzyme modify the properties of the whole drug?metabolizing ensemble in a complex, hard?to?predict manner. The current project is prompted by recognition of a far?reaching physiological significance and a critical pharmacological importance of these complex interrelationships in the P450 ensemble. Our central hypothesis is that the results of alcohol? induced increase in CYP2E1 content in human liver are not limited to direct consequences of the CYP2E1 activity, but also involve a multitude of alterations of the metabolism of various drugs and endogenous substrates caused by CYP2E1 interactions with other P450 species. These indirect effects of CYP2E1 expression are deeply involved in clinically? important cases of alcohol interactions with drugs and alcohol?induced alteration of P450?dependent signaling pathways. This proposal is aimed to probe the above hypothesis through a series of studies of the effects of changes in CYP2E1 content in human liver microsomes (HLM) on the system?wide properties of the drug? metabolizing system of human liver. The main model system chosen for this project is given by human liver microsomes where the composition of the P450 ensemble is modified by incorporation of purified recombinant human P450 enzymes, and CYP2E1 in particular. Specific Aims are: (1) To probe the impact of changing the content of ethanol?inducible CYP2E1 in human liver microsomes (HLM) on the profile of drug metabolism and the activity of the major drug?metabolizing cytochromes P450. (2) To identify the major partners of protein?protein interactions of ethanol? inducible CYP2E1 in the ER of human liver cells. The study is expected to provide a solid foundation for further exploration of CYP2E1?dependent interactions of alcohol with drugs (and the drugs of abuse, such as opioids, MDMA, and cocaine, in particular) both in cellular model systems and in vivo.